Ocular disease caused by herpes simplex virus affects up to 500,000 people in the United States. It is known that innate host resistance, host immune responses, and viral genes affect the severity of ocular disease. The continuing goal of this project has been to identify genes in HSV that are critical for the development of severe keratitis and to understand how the gene products contribute to disease severity. Several HSV genes are known to affect various components of virulence, including genes we have identified in past studies. Traditionally, approaches to such studies have focused on single genes at a time. In reality, multiple viral genes interact to promote virulence and little is known about such interactions and how they affect virulence. We have established a system with HSV whereby we can examine the contributions of multiple viral genes in the development of the virulence phenotype. Our system represents the first such system developed for a large DNA virus. Studies completed in the previous funding period have shown that there are at least six determinants involved. The US1 gene was identified as one determinant and several mutations were identified in the US1 gene. Using site directed mutagenesis, we have shown that reversion of either of two of the mutations (S34A and Y116C) restores virulence in marker transfer studies. The Specific Aims are to: (1) Construct viruses carrying reversions of the specific mutations and use these viruses for in vitro and in vivo studies on the effect of the specific mutations on viral growth, replication cycle events, ocular and neurovirulence, and latency; (2) test the hypothesis that S34 and Y116 are phosphorylation sites in the wild type HSV US1 protein using phosphoaminoacid analysis, antibody pulldowns and co-precipitation, and mass spectroscopy methods; (3) Test the hypothesis that specifically modified isoforms of the US1 protein carry out specific functions related to virulence and correlate isoforms with functions (e.g. isoform A is involved with function B). These studies will add to our knowledge of the role of the US1 protein in ocular HSV infections and may lead to improved therapies.